1. Summary of the original project Antibiotic resistance is a major threat to public health. An important mechanism of resistance to antibiotics such as tetracyclines is efflux mediated by membrane transporter proteins. The structural basis for substrate recognition by tetracycline efflux pumps, Tet proteins, is unknown. The efflux pump TetL from Bacillus subtilis exports tetracycline (Tc) in the form of a tetracycline-magnesium [Tc.Mg2+]+ complex, and is responsible for this bacterium?s resistance to the once widely efficacious antibiotic. TetL, with 14 transmembrane ?-helices, is a member of the family of Tet efflux proteins in Gram-positive bacterial pathogens, including Bacillus anthracis, Bacillus cereus, Streptococcus pneumoniae, Staphylococcus aureus. Clostridium spp., Enterococcus spp. and Listeria spp. All Tet transporters belong to the major facilitator superfamily (MFS). No crystal structure is available for any Tet protein or any MFS protein with 14 helices. A crystal structure of TetL, in combination with biochemical and biophysical studies, will not only greatly advance our understanding of the molecular mechanism of efflux-mediated antibiotic resistance, it will also suggest new ways to modify tetracycline to reverse resistance. 2. Major setback due to Sandy We will use nanobodies to improve diffraction resolution. Our experience with antibodies allowed us to rapidly progress in the evaluation of nanobodies produced through a collaboration with the Steyaert Lab in Brussels, Belgium. We have generated a panel of 18 nanobodies that recognize purified TetL. These nanobodies can be readily made and purified from bacterial hosts, providing a continuous supply of nanobodies for structural and functional studies. Of these, 12 produce a stable complex that has been used in co-crystallization studies. Crystals have been grown for 9 of these, with the best resolution reaching 4-5 . These TetL/nanobody complexes are being further optimized to improve diffraction resolution. Additionally, the nanobodies are being combined to form heteromeric complexes to increase the available crystallization space in order to facilitate higher resolution diffraction. These nanobodies may also bind to preferential conformations of TetL, which may structurally shed light on the conformational changes during the tetracycline transport cycle.